Commercial detergent protease preparations are commonly obtained by cultivation of selected strains of Bacillus licheniformis. The protease is elaborated extracellularly by the microorganism and a protease concentrate is subsequently recovered from the culture broth, e.g. by precipitation with salts and/or solvents. The work-up procedures may be conducted in such a manner that practically no protease activity is left in the culture broth. However, the recovery procedure is not particularly specific with respect to the protease, and in consequence, the protease concentrate will also normally contain broth constituents other than the enzyme. To produce a commercial detergent protease product, such as Alcalase supplied by Novo Industri A/S, Denmark (Alcalase being a Registered Trade Mark), the protease concentrate is then admixed with an inert filling material and the auxiliary agents which serve to produce a low dusting protease product of a predetermined proteolytic activity.
By far the most predominant proteolytic component of commercial Bacillus licheniformis derived protease products, such as Alcalase, has been identified as subtilopeptidase A (EC 3.4.21.14), hereinafter called subtilisin. This enzyme, which can be recovered from the commercial product in purified and crystallized form has been characterized in great detail and was found to belong to the same group of so-called serine proteases as trypsin and chymotrypsin. The enzyme nomenclature alludes to the fact that originally the microbial source of Alcalase had been classified as B. subtilis. However, it should now be generally accepted that the Alcalase producing microorganism is B. licheniformis.
Practically all proteins, including industrial enzymes, exhibit allergenic properties of varying potency, depending on the individual protein. A significant allergenic effect of Bacillus licheniformis derived protease preparations was experienced early after their commercial introduction and ever since then such allergenicity has been regarded by the art as an inherent inconvenience connected with the use of the protease products.
Heretofore, efforts to minimize the incidence of hypersensitive reactions among workmen and users have been directed solely towards providing low dusting particulate, e.g. granular or encapsulated, protease products so as to reduce the risk of exposure to the protease, both by workers in detergent enzyme and in washing powder manufacturing plants and by users of household washing powders. The success of these endeavours is, in part, evidenced by the continued widespread domestic use of detergent enzyme containing washing powders.
However, it is also known that the development of allergic reactions can be very unpredictable and capricious. Thus, there yet remains a need for a commercial protease preparation of allergenic properties substantially attenuated as compared with those preparations known heretofore.
It is an object of the present invention to provide a B. licheniformis protease product attenuated in allergenicity.
It is another object of the present invention to provide an industrially feasible process for preparing a product having reduced allergenicity.
The attainment of these objects is based on certain observations relating to the constituents of commercial protease preparations and to their properties.
Recently published investigations by means of qualitative (but highly sensitive), immunoelectrophoresis (e.g. according to Grabar-Williams, vide R. Verbruggen et al. Biochim, Biophys, Acta, vol. 365 (1974), pp. 108-114) indicated that commercially available protease products, notably Alcalase are antigenically heterogeneous.
In subsequent studies published by Verbruggen (Biochem. Journal, vol. 151 (1975), pp. 149-155), using the technique of quantitative crossed agarose gel immunoelectrophoresis, it was found that the main protease component of Alcalase, consisting of a family of subtilisin isoenzymes, is accompanied by a minor protein component, which is antigenically different from the main protease component.